ABC technique with Western blot - Do you get extra band with ABC? (Mar/18/2005 )

I am using biotinylated anti-rat for my secondary antibody and subsequently need to incubate the membrane in ABC for an hour before developing using the ECL. My primary antibody is monoclonal and hence should be very specific.

Question: Anyone out there knows about the likelihood of getting extra band apart from the band of interest. Would the problem be completely eradicated using horse radish peroxidase (HRP) anti-rat as my secondary antibody.

All comments would be welcome.

Cheers

Hi! I have the same problem as you have, and I tried different combinations of antibodies, and even the ABC complex alone and always obtain two nice bands from the ABC complex. I think that if you have the possibility of using a HRP conjugated antibody this problem should be solved. Good luck
cheers